Peptide-based inhibitors of HIV replication

ABSTRACT

Described herein are oligopeptides useful to inhibit replication in virally infected individuals. In a preferred embodiment of the invention, the oligopeptide is a D-arginine nonamer having N- and C-terminal protecting groups, which, at a 5 uM concentration, exhibits greater than 95% inhibition of HIV replication, in a standard assay.

This application is a continuation of application Ser. No. 07/779,735,filed Oct. 23, 1991, abandoned, which is a continuation-in-partapplication of Ser. No. 07/602,953 filed on Oct. 24, 1990, nowabandoned.

After integrating into its human cell host, the human immunodeficiencyvirus, HIV, undergoes highly controlled stages of replication. Infectionof an individual is followed initially by a phase, known as the latencyperiod, in which viral replication is either non-existent or occurs atonly a very low, basal level. In a later phase however, which marksprogression of the acquired immune deficiency syndrome or AIDS, theviral replication rate is remarkably accelerated, and leads ultimatelyto cell death and the continued spread of infection. Studies have shownthat one of the principle mediators of accelerated viral replication isan HIV-produced protein know as tat.

While select strains of HIV produce different forms of tat, a 72 aminoacid N-terminal sequence is common to all forms. The principle form oftat, herein designated tat(1-86), consists of 86 amino acids arranged inthe sequence reported by Ratner et al., 1985, Nature 313:277,incorporated herein by reference. Structure/function analysis of thevarious tat forms has revealed at least three domains in the protein,including a proline-rich region spanning residues 1-18, a cysteine-richregion spanning residues 22-37, and a basic region of nine amino acidspanning residues 49-57.

Following accumulation in its cellular environment, tat elicits adramatic increase in the rate at which gene products essential to HIVreplication are expressed, a process which is referred as a"transactivation". Recent studies demonstrate that tat's ability toamplify gene product formation is mediated through its bindinginteraction with an RNA hairpin structure, known as the "transactivatorresponsive element" or TAR element, borne at the 5'end of all HIV genetranscripts (see for example Weeks et all, infra). It has accordinglybeen suggested that agents capable of interfering with the interactionbetween tat protein and the RNA TAR element could inhibit viralreplication, and thus be useful therapeutically to treat virallyinfected individuals. In this area of research, some effort is aimed atidentifying transactivation-deficient analogues of tat that antagonizetat action by competing with it for binding to the TAR element.

Variants of tat(1-86) have been generated by the expression ingenetically engineered microbial hosts of an appropriately mutagenizedclone of the HIV tat gene (see example Kuppuswamy et al, Nucl. AcidsRes., 1989, 17(9): 3551). These variants have been useful in identifyingessential structural features and in mapping functional domains of tat.Kuppuswamy et al found, for example, that tat analogues bearingsubstitutions in the cysteine-rich domain of tat (residues 22-37) werealmost totally defective in transactivation, and postulated that thecysteine residues were essential for tat activity. Also defective intransactivation were tat analogues lacking the basic domain (residues49-57). The authors suggest that the basic domain is important also celluptake of tat, and for efficient transport of tat to the nucleus.

Mutational analysis of the basic domain of tat has been reported also byHauber et al in J. Virol., 1989, 63(3): 1181, who showed that thetransactivation function of tat is markedly reduced when positivelycharged amino acids in the basic domain of tat(1-86) are replaced byneutral amino acid residues.

Green et al have also studied the transactivation function of varioustat fragments (see Cell, July 1989, 58:215). While an oligopeptiderepresenting tat residues 37-72 was found to mediate transactivation,variants thereof bearing substitutions at residues 41, 46 or 47displayed negligible transactivation, and are described by the authorsas potential antagonists of tat action (see also Green et al, inWO89/12461 published 28 Dec. 1989). Weak tat antagonist activity wasalso detected in an oligopeptide representing the basic domain of tati.e. ⁴³ Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg⁵⁷.

Weeks et al. in a highly refined study, have confirmed that tat doesindeed bind to the HIV TAR element, and have pointed more specificallyto a binding interface between the tat basic domain and a threenucleotide bulge extending from the stem of the TAR hairpin (seeScience, 1990, 249:1281).

It is a general object of the present to provide a compound useful toinhibit replication of the immunodeficiency virus, HIV.

It is a more specific object of the present invention to provide acompound capable of inhibiting tat-mediated transactivation of HIV geneexpression.

It is another object of the present invention to provide apharmaceutical composition useful to inhibit replication of HIV.

SUMMARY OF THE INVENTION

It has now been found that the tat antagonist properties of that tatbasic domain can be improved through structural modification thereof. Ithas more specifically been found that alterations in the amino acidsequence constituting the tat basic domain can confer enhancedTAR-binding affinity, thus providing compounds better able to competewith endogenous tat for binding to TAR. It has further been found thatthe TAR-binding affinity of the tat basic domain is not substantiallycompromised when the L-amino acids which occur naturally in its sequenceare replaced with the less protease-sensitive D-amino acids.Advantageously, none of the modifications herein described alter thehighly basic amino acid composition responsible for both cellular uptakeand nuclear localization of tat. Moreover, the tat basic domainanalogues of the present invention bind to the HIV TAR element with aselectivity similar to that exhibited by tat.

More particularly, and according to one aspect of the invention, thereis provided a transactivation-deficient, HIV TAR-binding compound of theformula:

    R1--(A)m--[X]--(B)n--R2

wherein

R1 is H or an N-terminal protecting group;

R2 is OH or a carboxyl terminal protecting group;

X represents a TAR-binding, transactivation-deficient oligopeptideanalogue of the tat basic domain, consisting of from 7 to 12amide-linked α-amino acid residues;

m is 0 or 1;

n is 0 or 1; and

A and B independently represent one or more amide-linked, α-amino acidresidues which collectively are selected to retain thetransactivation-deficient nature of the compound.

According to one embodiment of the invention, X in the above formularepresents an oligopeptide having a TAR binding affinity that isenhanced relative to HIV tat. According to another embodiment of thepresent invention, X in the above formula represents an oligopeptidecomprising at least one D-amino acid, and more desirably consistsessentially of D-amino acids. A preferred compound of the presentinvention consists of nine D-arginine residues having blocking groups atboth the N- and C-termini.

According to another respect of the present invention, there is provideda pharmaceutical composition comprising a transactivation-deficient,TAR-binding compound of the invention, and a pharmaceutically acceptablecarrier.

According to another aspect of the present invention, there is provideda method for treating a patient infected with HIV, which comprisesadministering to the patient an effective amount of a compound of thepresent invention.

The terms "amino acid" and "α-amino acid residue" are usedinterchangably herein with reference to naturally occurring andsynthetic amino acids in either D- or L-form. Unless otherwise stated,the amino acid is the naturally occurring L-amino acid. Included, unlessotherwise stated, are are the amino acids glycine; those amino acidshaving an aliphatic α-carbon side chain such as alanine, valine,norvaline, leucine, norleucine, isoleucine and proline; those havingaromatic α-carbon side-chains such as phenylalanine, tyrosine andtryptophan; those having acidic α-carbon side chains such as asparticacid and glutamic acid; those having side chains which incorporate ahydroxyl group such as serine, homoserine, hydroxynorvaline,hydroxyproline and threonine; those having sulfur-containing α-carbonside chains such as cysteine and methionine; those having side chainsincorporating an amide group such as glutamine and asparagine; and thosehaving basic α-carbon side chains such as lysine, arginine, histidine,and ornithine (also herein referred to as "basic amino acids").

The term "N-protecting group" refers to a radical attached to thenitrogen atom which serves to protect the amino terminus of theoligopeptide from undesired biochemical attack.

The term "carboxyl protecting group" refers to a radical attached to theC-terminus of the oligopeptide either via an oxygen or via the carbon ofthe terminal carboxyl group, which serves to protect the carboxylterminus of the oligopeptide from undesired biochemical attack.

BRIEF REFERENCE TO THE DRAWINGS

FIG. 1 illustrates graphically the results of HIV inhibition withcompounds of the present invention.

DETAILED DESCRIPTION OF THE INVENTION AND ITS PREFERRED EMBODIMENTS

The compounds of the invention are transactivation-deficient,TAR-binding oligopeptides, which function as antagonists of tat action.

The terms "oligopeptide" and "polypeptide" are used interchangablyherein with reference to a compound having from about 6 to about 100 ormore amide-linked α-amino acid residues.

The term "transactivation" refers to the tat-mediated enhancement of HIVgene expression, which results in elevated viral mRNA level andactivity, probably as a result of tat-mediated effects on transcriptionfrom the viral long terminal repeat (LTR--which includes the sole viralpromoter) and perhaps on tat-mediated effects on translation. Thetransactivation effect of a given compound can be determined directly byincubating the compound with HIV-infected cells and then measuring theincrease in viral plaque formation, or the increase in production of oneor more viral proteins, such as p24. Assays ore convenient for measuringtransactivation have been developed and, because they do not require useof intact virus, are now more commonly used. In these assays, acandidate compound is incubated with mammalian cells harbouringrecombinant DNA cassettes in which DNA coding for a convenientlydetectable marker protein is placed under the expression control of theHIV LTR. Formation of marker protein following uptake of the candidatecompound is thus a direct measure of its ability to mediatetransactivation. An example of one such convenient transactivationassay, which employs a bacterial chloramphenicol transferase (CAT) asthe marker protein, is described by Ruben et al in J. Virol., 1989,63(1): 1.

The term "transactivation deficient" used herein to characterizecompounds of the invention thus refers to oligopeptides and polypeptideswhich following cell uptake are incapable of mediating a statisticallysignificant increase in LTR-mediated gene expression, as measured by anyone of the conventionally used means for detecting transactivation.

The term "TAR-binding", used herein to characterize compounds of theinvention, refers to compounds which exhibit the ability to bind the HIVTAR element, as determined by an RNA mobility shift electrophoresisassay (see for example Roy et al, infra). This assay generally involvesthe incubation of a selected oligopeptide with gel-purified,radio-labelled TAR RNA, or tat-binding fragment of TAR RNA, that hasbeen produced either by direct synthesis or through transcription of acorresponding DNA template. Samples are loaded onto non-denaturingpolyacrylamide gel and subjected to electrophoresis, and then migrationof labelled RNA through the gel is visualized on gel-exposed, X-rayfilm. Oligopeptides which bind TAR are identified in this assay by theircharacteristic, retarded migration on the gel, relative to TAR RNA towhich no oligopeptide has bound. In this mobility shift assay,oligopeptides having an "enhanced" affinity for binding TAR RNA willresolve on the gel as bands which, in addition to displaying theretarded migration pattern characteristic of TAR-binding oligopeptides,will also appear with greater intensity and/or size than a bandcontaining TAR RNA bound to tat (1-86).

The term "basic domain" refers to the basic domain of the HIV tatprotein, which is an oligopeptide consisting of nine L-amino acidsarranged in the sequence Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg.Oligopeptides which differ from the basic domain in terms of amino acidsequence, either by way of addition, deletion or replacement of one ormore amino acids including replacement of an L-amino by a correspondingD-amino acid, are referred to herein as "analogues" of the tat basicdomain.

In one of its aspects, the present invention provides Tar-binding,transactivation deficient compounds which conform to the general formula(I)

    R1--(A)m--[X]--(B)n--R2                                    (I)

wherein:

X represents a Tar-binding, transactivation deficient oligopeptideanalogue of the basic domain of the HIV tat protein i.e. an analogue ofthe amino acid sequence Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg, whichconsists of from 7 to 12 amide-linked α-amino acids;

A represents one or more amide-linked, α-amino acids coupled to theN-terminus of X;

B represents one or more amide-linked, α-amino acids coupled to theC-terminus of X, with the proviso that both A and B are selected suchthat the compound of formula I exhibits both Tar-binding affinity andtransactivation-deficiency;

m and n are, independently, either 0 or 1;

R1 represents H or an N-protecting group (Np); and

R2 represents OH or a carboxyl-protecting group (Cp).

Preferred compounds of the present invention are those of formula (Ia),

    R1--[X]--R2                                                (Ia)

in which R1, R2 and X are as specified above.

Particularly preferred are compounds of formula (Ib),

    Np--[X]--Cp                                                (Ib)

in which X is as specified above, Np represents an N-terminal protectinggroup, and Cp represents a carboxyl terminal protecting group.

In each of the above formulae I, Ia and Ib, X represents an oligopeptideconsisting of from 7 to 12 amide-linked amino acid residues, preferably8 to 10 amino acid residues, arranged in a sequence that confersTAR-binding affinity. According to a preferred aspect of the presentinvention, X comprises at least one amino acid in the D-isomer form. Theoligopeptide X may, for example, comprise alternating L- and D-aminoacids. Most preferably the oligopeptide consists essentially of D-aminoacids. With respect to amino acid composition, X in each of the aboveformulae I, Ia and Ib is an oligopeptide analogue of the HIV tat basicdomain desirably having a net positive charge of at least n-1, where nrepresents the number of amino acids constituting the oligopeptide X.With respect to composition and sequence, X is selected desirably fromamong the group consisting of;

i) an oligopeptide having the sequence

Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg, wherein at least one amino acid isa D-amino acid;

ii) an oligopeptide consisting of from 6 to 11 basic amino acids and oneamino acid other than a basic amino acid, wherein each basic amino acidis independently selected from among the group consisting of arginine,lysine, histidine and ornithine, and said one amino acid is selectedfrom among the group consisting of glutamine, serine, histidine, lysine,asparagine and homoglutamine. Especially suitable oligopeptides arethose in which each basic amino acid is independently selected fromarginine and lysine, and the non-basic amino acid is glutamine; and

iii) an oligopeptide consisting essentially of from 7 to 12 basic aminoacids, wherein each basic amino acid residue is independently selectedfrom among the group consisting of lysine and arginine.

According to specific embodiments of the present invention, X representsan oligopeptide selected from among the group consisting of:

iv) an oligopeptide comprising amino acids arranged in the sequenceArg-Lys-Lys-Arg-Arg-Y1-Arg-Arg-Arg, wherein Y1 is a basic amino acid;

v) an oligopeptide comprising amino acids arranged in the sequenceArg-Y2-Y3-Arg-Arg-Y4-Arg-Arg-Arg wherein each of Y2, Y3 and Y4 is abasic amino acid, and at least one of Y2, Y3 and Y4 is arginine;

vi) an oligopeptide comprising from 6 to 11 arginines and one glutamine;and

vi) an oligopeptide homopolymer consisting of from 7 to 12 arginines.

According to preferred embodiments of the present invention, X in theabove formula I, Ia and Ib represents an oligopeptide, preferablyconsisting essentially of D-amino acids, having an amino acid sequenceselected from: ##STR1##

Particularly preferred compounds of the present invention are those offormula I, Ia and Ib in which X represents either homopolymericD-arginine, having 8, 9 or 10 amide-linked D-arginine residues, or anoligopeptide comprising one at least one D-Gln residue and 7, 8 or 9D-Arg residues. The presently most preferred compounds are those whereinX in the above formulae I, Ia and Ib represents an oligopeptideconsisting essentially of D-amino acids and having an amino acidsequence selected from: ##STR2##

As noted hereinabove, the compounds of the present invention aredesirably those of formula (Ib)

    Np--[X]--Cp                                                (Ib)

wherein X represents an oligopeptide as defined above, Np represents anN-terminal protecting group, and Cp represents a carboxyl terminalprotecting group. Any chemical group which serves to protect peptideends from undesired chemical attack can be used. Carboxyl terminalprotecting groups and N-terminal protecting groups employedconventionally in the art of peptide synthesis are most desirablyincorporated in the compounds of the present invention. UsefulN-terminal protecting groups include, for example, loweralkanoyl groupsof the formula R--C(O)-- wherein R is a linear or branched lower alkylchain comprising from 1-5 carbon atoms. A preferred N-terminalprotecting group is acetyl, CH₃ C(O)--. Also useful as N-terminalprotecting groups are amino acid analogues lacking the amino function.

Preferred C-terminal protecting groups are, similarly, those usedconventionally in the art of peptide synthesis. Such C-terminalprotection may be achieved by incorporating the blocking group via thecarbon atom of the carboxylic function, for example to form a ketone oran amide, or via the oxygen atom thereof to form an ester. Thus, usefulcarboxyl terminal protecting groups include, for example, ester-formingalkyl groups, particularly lower alkyl groups such as e.g., methyl,ethyl and propyl, as well as amide-forming amino functions such asprimary amine (--NH2), as well as monoalkylamino and dialkylamino groupssuch as methylamino, ethylamino, dimethylamino, diethylamino,methylethylamino and the like. C-terminal protection can also beachieved by incorporating as the C-terminal amino acid a decarboxylatedamino acid analogue, such as agmatine. Of course, N- and C-protectinggroups of even greater structural complexity may alternatively beincorporated, if desired.

Especially preferred compounds of the invention, which conform toformula (Ib), are acetyl-[(D-Arg)₉ ]--NH₂ ; acetyl-(D-Arg)₃--(D-Gln)--(D-Arg)₅ --NH₂ ; andacetyl-[D-(Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg)]-NH₂.

It will be appreciated that the oligopeptide may be conjugated, eitherthrough its C-terminus or its N-terminus to other amino acids withoutnecessarily sacrificing the characteristic transactivation-deficiencyand Tar-binding property exhibited by the oligopeptide, as determined bythe assays herein described. The present invention thus further embracesTar-binding, transactivation deficient polypeptide compounds whichincorporated the oligopeptides described herein and conform to thegeneral formula (I), i.e.

    R1--(A)m--[X]--(B)n--R2                                    (I)

wherein at least one of m and n is 1, A and B independently representone or more amide-linked, alpha amino acids, and R1, R2 and X are asspecified above. Desirably, R1 represents an N-terminal protectinggroup, Np, and R2 represents a carboxyl terminal protecting group, Cp,wherein Np and Cp are as defined above.

Specifically contemplated compounds of formula I aretransactivation-deficient fragments and variants of HIV tat in which theresident basic domain (residues 49-57) is replaced by an oligopeptide ofthe present invention. It has been shown, for example, that variants oftat(37-62) in which alanine substitutions are introduced at positionsflanking the basic domain e.g. positions 41, 46 and/or 47 exhibit strongantagonist activity (Green et al, supra). Specifically contemplated arecompounds in which the basic domain of such tat(37-62)-based antagonistsis replaced by an oligopeptide of the present invention. Most desirably,amino acids representing A and B in the above formula I are D-aminoacids.

The compounds of the present invention can be readily prepared bystandard, well-established solid-phase peptide synthesis methods (SPPS),general descriptions of which appear, for example, in J. M. Stewart andJ. D. Young, Solid Phase Peptide Synthesis, 2nd Edition, 1984, PierceChemical Company, Rockford, Ill.; and in M. Bodanszky and A. Bodanszky,The Practice of Peptide Synthesis, 1984, Springer-Verlag, New York;Applied Biosystems 430A Users Manual, 1987, ABI Inc., Foster City,Calif.

In general, a suitably protected amino acid is attached through itscarboxyl group (--COOH) to a derivatized, insoluble polymeric support,e.g. cross-linked polystyrene or polyamide resin. "Suitably protected"refers to the presence of protecting groups on the alpha-amino group(α-NH₂) and side-chain functional group (if present) of the amino acid.Synthesis proceeds in a stepwise, cyclical fashion by successivelyremoving the α--NH₂ protecting group, then coupling an activated aminoacid to the newly freed α-NH₂. Activation of the --COOH group of theincoming amino acid can be effected directly via a carbodiimide, e.g.dicyclohexylcarbodiimide (DCC) or diisopropylcarbodiimide (DIC), viaformation of the symetric acid anhydride, or preferably by formation ofan "active ester", e.g. hydroxybenzotriazole (HOBt), pentafluorophenyl,para-nitrophenyl or N-hydroxysuccinimide esters. Suitable side-chainprotecting groups generally are stable to all of the reagents, solventsand reaction conditions used during synthesis, yet removable underconditions which will not affect the integrity of the final peptideproduct.

The two preferred methods of solid phase peptide synthesis are the BOCand FMOC methods, so called for their use of the tert-butyloxycarbonyland 9-fluorenylmethyloxycarbonyl groups, respectively, to protect theα-NH₂ of the amino acid residues.

In the more established BOC method, the acid-lability of the BOC groupis exploited and trifluoroacetic acid (TFA) treatment is used to effectits removal. The preferred amino acid side-chain protecting groups (forexamples see Table 1 below) are relatively stable in weak acid, e.g.TFA. Most can be cleaved by very strong acids such as hydrofluoric (HF)or trifluoromethanesulfonic acid (TFMSA). A few side-chain protectinggroups, e.g. His(Dnp) & Met(O), may require a separate deprotectionstep, e.g. thiophenol or ammonolysis, mercaptopyridine ormercaptoethanol treatment, respectively. After synthesis, the peptide istypically cleaved from the resin and simultaneously deprotected by HFtreatment at low temperature, e.g. 0° C.

                  TABLE 1                                                         ______________________________________                                        Examples of Side-Chain Protecting Groups Used in                              SPPS by the BOC Method                                                                  Side-Chain                                                          Residue   Moiety    Protecting Group                                          ______________________________________                                        Arginine  guanidino p-toluenesulfonyl (Tos);                                                      methoxybenzenesulfonyl (Mts);                                                 nitro.                                                    Aspartic Acid,                                                                Glutamic Acid                                                                           carboxyl  ortho-benzyl (OBz1)                                       Cysteine  sulfhydryl/                                                                             p-methylbenzyl (CH.sub.3 Bzl)                                       thiol                                                               Histidine imidazole 2,4-dinitrophenyl (Dnp); (Tos)                                      N-H                                                                 Lysine    amino     2-chlorobenzyloxycarbonyl (Cl-z)                          Methionine                                                                              sulfide/  sulfoxide (O); none                                                 thioether                                                           Serine,                                                                       Threonine hydroxy   benzyl (Bzl)                                              Tryptophan                                                                              indole N-H                                                                              formyl (CHO)                                              Tyrosine  hydroxy   2-bromobenzyloxycarbonyl (Br-z)                           ______________________________________                                    

In the more recently developed FMOC method the base labile FMOC group isremoved using a mild organic base, e.g. piperidine, thereby allowing theuse of side-chain protecting groups which are labile to milder acidtreatment, e.g. TFA (for examples see Table 2). An acid labile etherresin such as HMP-resin (para-hydroxymethylphenoxymethyl polystyrene) isused as the solid support, permitting simultaneous cleavage/deprotectionin TFA.

                  TABLE 2                                                         ______________________________________                                        Examples of Side-Chain Protecting Groups Used in                              SPPS by the FMOC Method                                                                 Side Chain                                                          Residue   Moiety      Protecting Group                                        ______________________________________                                        Arginine  guanidino   4-methoxy-2,3,6-trimethyl-                                                    benzenesulfonyl (Mtr);                                                        pentamethylchroman-6-sulfonyl                           Aspartic Acid,                                                                Glutamic Acid                                                                           carboxy     t-butyl ester (OtBu)                                    Cysteine  sulfhydryl/ trityl (Trt);                                                     thiol                                                                                     acetamidomethyl (Acm)                                   Histidine imidazole N-H                                                                             Trt                                                     Lysine    amino       t-butyloxycarbonyl (BOC)                                Serine,                                                                       Threonine,                                                                    Tyrosine  hydroxyl    t-butyl (tBu)                                           ______________________________________                                    

Suitably protected and/or preactivated D- and/or L-amino acids,derivatized and/or preloaded resins, and all ancillary reagents andsolvents required for either BCC or FMOC peptide synthesis arecommercially available from several suppliers. In addition, automatedpeptide synthesizers with optimized, pre-programmed BOC and/or FMOCsynthesis cycles are available from numerous commercial sources.

Incorporation of N- and/or C-protecting groups can also be achievedusing protocols conventional to solid phase peptide synthesis methods.For incorporation of C-terminal protecting groups, for example,synthesis of the desired peptide is typically performed using, as solidphase, a supporting resin that has been chemically modified so thatcleavage from the resin results in a peptide having the desiredC-terminal protecting group. To provide peptides in which the C-terminusbears a primary amino protecting group, for instance, synthesis isperformed using a p-methylbenzhydrylamine (MBHA) resin so that, whenpeptide synthesis is completed, treatment with hydrofluoric acidreleases the desired C-terminally amidated peptide. Similarly,incorporation of an N-methylamine protecting group at the C-terminus isachieved using N-methylaminoethyl-derivatized DVB resin, which upon HFtreatment releases peptide bearing an N-methylamidated C-terminus.Protection of the C-terminus by esterification can also be achievedusing conventional procedures. This entails use of resin/blocking groupcombination that permits release of side-chain protected peptide fromthe resin, to allow for subsequent reaction with the desired alcohol, toform the ester function. FMOC protecting groups, in combination with DVBresin derivatized with methoxyalkoxybenzyl alcohol or equivalent linker,can be used for this purpose, with cleavage from the support beingeffected by TFA in dicholoromethane. Esterification of the suitablyactivated carboxyl function e.g. with DCC, can then proceed by additionof the desired alcohol, followed by deprotection and isolation of theesterified peptide product.

Incorporation of N-terminal protecting groups can be achieved while thesynthesized peptide is still attached to the resin, for instance bytreatment with suitable anhydride and nitrile. To incorporate an acetylprotecting group at the N-terminus, for instance, the resin-coupledpeptide can be treated with 20% acetic anhydride in acetronitrile. TheN-protected peptide product can then be cleaved from the resin,deprotected and subsequently isolated.

Once the desired peptide sequence has been synthesized, cleaved from theresin and fully deprotected, the peptide is then purified to ensure therecovery of a single oligopeptide having the selected amino acidsequence. Purification can be achieved using any of the standardapproaches, which include reversed-phase high-pressure liquidchromatography (RP-HPLC) on alkylated silica columns, e.g. C₄ -, C₆ -,or C₁₈ -silica. Such column fractionation is generally accomplished byrunning linear gradients, e.g. 0-50%, of increasing % organic solvent,e.g. acetonitrile, in aqueous buffer, usually containing a small amountof TFA, e.g. 0.1%. Alternatively, ion-exchange HPLC can be employed toseparate peptide species on the basis of their charge characteristics.Column fractions are collected, and those containing peptide of thedesired/required purity are pooled together. The peptide is typicallythen treated to exchange the cleavage acid (e.g. TFA) with apharmaceutically acceptable acid, such as acetic acid, to provide awater soluble salt of the peptide.

Following purification, it is desirable to analyze the oligopeptidefurther to ensure its chemical authenticity. This is most convenientlyachieved through amino acid composition analysis. To analyze amino acidcomposition, a sample of purified oligopeptide is completely hydrolysedin aqueous acid, e.g. HCl, and the resulting mixture of amino acidsseparated, identified and quantitated via HPLC, e.g. Waters Pico-Tagsystem, or automated analyzer, e.g. Beckman 6300 Amino Acid Analyzer. Amore definitive measure of authenticity is full sequence analysis of thepeptide. Several protein sequenators which sequentially degrade thepeptide and identify the linear order of its amino acids are used forthis purpose, and are available from several commercial sources.High-resolution mass spectrometry methods can also be applied, togenerate exact molecular weight information.

For therapeutic use, the oligopeptide compounds of the invention aredesirably of "pharmaceutical grade" purity, a term used herein withreference to an oligopeptide preparation which has been shown to migrateas a single peak on HPLC, to exhibit uniform and authentic amino acidcomposition and sequence upon analysis thereof, and which otherwisemeets standards set by the various national bodies which regulatequality of pharmaceutical products.

For therapeutic use, compounds of the present invention exhibitingpharmaceutical grade purity are combined with pharmaceuticallyacceptable carriers to generate compositions suitable for administrationto patients. Any of the carriers conventionally used in thepharmaceutical industry may be employed, such as diluents, excipientsand the like. According to a preferred embodiment of the invention, thecompounds are formulated for administration by injection, eithersub-cutaneously or intravenously, and are accordingly provided asaqueous, buffered compositions, in sterile and pyrogen-free form. Thecompounds herein designated as preferred compounds are substantiallywater-soluble. Water solubility of these and other compounds of theinvention may be enhanced, if desired, by incorporating a solubilityenhancer, such as cetyltrimethylammonium bromide or chloride.Alternatively, the compounds of the present invention may be formulatedfor administration by routes other than injection, of course.Compositions for topical application, such as creams, lotions orointments can be used, as may aerosol inhalable formulations. Oraldosage forms, such as tablets, capsules and the like, formulated inaccordance with standard pharmaceutical practise, may also be employed.

Pharmaceutical compositions containing a compound of the presentinvention are useful to treat HIV-infected patients, i.e. patientsdiagnosed as being carriers of the HIV virus. The compounds of theinvention are most desirably administered to HIV-infected patientsbefore clinical symptoms of the AIDS syndrome are manifest, as a meansof delaying and/or arresting progression of the syndrome. Suitabletreatment regimens are those which maintain in the patient a serum levelof the compound sufficient to control HIV replication. The establishmentin the patient of such effective levels will be reflected, inappropriately controlled trials, by a reduction in the rate at which T4lymphocytes are depleted, or by the absence in patient plasma samples ofan increase in HIV antigens, such as p24.

It is anticipated that an effective treatment regimen will entailintravenous administration of dosages sizes in the range from lug toabout 10 mg per kg, and such as between about 10 ug/kg to about 5 mg/kg.For example, it is anticipated that the peptide designated herein aspeptide 4C i.e. acetyl-[D-Arg₉ ]-NH₂, will be effective when present inserum at a concentration between about 1 uM and 100 uM, and that thisserum concentration can be achieved through maintenance doses in thesize range from about 0.1 mg/kg to about 5 mg/kg. It will be appreciatedhowever, that effective dosage sizes will vary according to the route ofadministration, and the frequency of administration. For example,smaller doses may suffice if administered on a daily basis, and largerdoses may be required if administration frequency is extended, or if thecompound is administered by a route other than intravenous injection.

EXAMPLES

To evaluate the effect of amino acid substitution in the tat basicdomain on TAR-binding affinity and specificity, oligopeptide variantsthereof were synthesized using the solid phase synthesis approach, andthen purified to homogeneity as measured by various means including highperformance liquid chromatography (HPLC). Amino acid compositionanalysis was employed to confirm authenticity of all oligopeptides,except the arginine homopolymer the chemical authenticity of which wasconfirmed by fast atom bombardment mass spectroscopy.

TAR-binding affinity of the oligopeptides relative to HIV tat(1-86) andrelative to tat fragments representing the tat basic domain, wasevaluated by RNA gel mobility shift assay using as ligand a truncatedfunctional form of TAR RNA having the sequence shown below. Forcomparison, the binding affinity for a variant of TAR which does notbind tat, was also assayed in the same manner, to evaluate thespecificity for TAR. ##STR3##

Synthesis of the TAR fragment and its variant analogue was accomplishedas described by Roy et al. in Genes and Development, 1990, 4: 1365. TheTAR-binding assay was also performed in the manner reported by Roy etal., supra, which is incorporated herein by reference.

EXAMPLE 1 Effect of Glutamine Substitution on TAR-Binding Affinity

To assess the contribution to TAR-binding of the glutamine residueinternal to the tat basic domain, a series of glutamine-substitutedanalogues was synthesized and then assayed in the RNA gel mobility shiftassay for TAR-binding activity.

Each of the oligopeptides was synthesized by, and purchased from, theAmerican Peptide Company, using the solid phase peptide synthesisapproach, and in accordance with protocols conventional thereto. Moreparticularly, synthesis was performed on a Beckman 990 synthesizer,using chloromethyl-polystyrene as solid support, and Boc-based protocolsand protecting groups, to generate the following compounds; ##STR4##

The TAR-binding affinity was then evaluated in the RNA gel mobilityshift assay described hereinabove, and compared with tat (1-86) and thecorresponding wild type oligopeptide. Results indicated thatsubstitution of glutamine by glycine and cysteine reduced TAR-bindingaffinity whereas oligopeptides in which glutamine was replaced byserine, histidine, asparagine or homoglutamine each displayed anaffinity for TAR similar to tat(1-86) and an oligopeptide representingthe basic domain thereof. Surprisingly, however, substitution ofglutamine by lysine resulted in an oligopeptide which displayed fargreater affinity for binding with TAR.

EXAMPLE 2 Evaluation of the Positional Significance of Gln⁵⁴

To investigate the positional significance of the glutamine residue inthe tat basic domain, a series of analogues was designed in which theposition of the glutamine varied in the context of an otherwisehomopolymeric L-arginine nonamer. Oligopeptides in this series were alsopurchased from American Peptide Company, and were synthesized by BOCchemistry and purified using conventional procedures, to yield theacetate salt of the following compounds: ##STR5##

TAR RNA binding assays revealed that each oligopeptide in this seriesbound TAR with affinity comparable to both tat (1-86) and theoligopeptide representing its basic domain. Each oligopeptide furtherdisplayed a specificity for binding to the functional TAR fragment asdetermined by the absence of substantial binding to the TAR variant.

EXAMPLE 3 Synthesis of L-(Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg)

With the results of examples 1 and 2 suggesting that the internalglutamine residue can be sacrificed, synthesis and evaluation of anL-Arg nonamer were undertaken. Nona-L-arginine, (L-Arg), was prepared bythe BOC solid-phase synthesis method. Synthesis was performed by TheAmerican Peptide Company using a Beckman 990 synthesizer andchloromethylpolystyrene resin as solid support.

The tert-butyloxycarbonyl group (BOC) was used to protect the α-NH₂function of L-arginine during the synthesis. The guanidino function wasprotected with the para-toluenesulfonyl group (Tos). Couplings werecarried out using excess hydroxybenzotriazole (HOBt)-activated ester ofBOC-L-Arg(Tos). Removal of the BOC protecting group after each cycle waseffected with TFA. The final peptide, (L-Arg)₉ was cleaved from thepolymer resin and the Tos protecting groups removed via standard HFtreatment. After removal of HF, the peptide+resin mixture was washedwith diethyl ether and extracted with aqueous acetic acid.

The crude peptide was lyophilized, then fractionated by RP-HPLC on a C₁₈silica column using a gradient of 2-40% acetonitrile in 0.1% TFA.Fractions were collected and checked by analytical RP-HPLC. Thosecontaining ≧95% of the major product were combined. High resolution massspectrometry showed the product to be the expected L-(Arg)₉.

The TAR RNA binding studies revealed that the arginine homopolymer boundwith greater affinity to TAR RNA than either tat (1-86), theoligopeptide representing the basic domain thereof, or any one of theoligopeptide analogues described in examples 1 and 2. The argininehomopolymer also bound selectively to the functional TAR fragment.

EXAMPLE 4 Synthesis of D-(Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg)

The named D-peptide is readily prepared by the FMOC solid-phasesynthesis method and an automated synthesizer, e.g. Applied Biosystems430A.

α-Amino groups of the D-amino acids are protected with the base-labilefluorenylmethyloxycarbonyl group (FMOC). The lysine and arginineside-chains are protected via acid-labile protecting, e.g. BOC anmethoxytrimethylbenzenesulfonyl (MtR), respectively.

The C-terminal FMOC-D-Arg(Mtr) residue is double-coupled to a suitablyderivatized polystyrene resin, e.g. HMP-polystyrene, via the symmetricanhydride. Removal of the FMOC group is carried out in 20% piperidine.Addition of amino acid residues to the peptide-resin is effected viatheir activated HOBt esters.

Cleavage and deprotection of the final peptide is carried out bytreatment with TFA. The crude peptide is purified by RP- or ion exchangeHPLC. The purified product is characterized by standard amino acidanalysis and/or mass spectrometry and/or sequence analysis.

A purified form of the named D-oligopeptide synthesized as describedabove was purchased from a commercial source, and tested in the TAR RNAbinding assay. The results of the assay revealed binding of theoligopeptide to the RNA, indicating that Tar binding is retained byoligopeptides in which L-amino acids are replaced by the correspondingD-amino acids.

EXAMPLE 5 Synthesis of Acetyl-[D-Arg]₉ -NH₂

The title compound, designated compound 4C, was synthesized usingp-methylbenzhydrylamine (MBHA) resin as solid support, to provide theC-terminal blocking amine on the resultant peptide. Synthesis proceededusing D-arginine residues in which the amino function was blocked withthe t-BOC group, and the guanidino function was blocked with the Tosgroup. Coupling cycles and deprotection were performed as described forthe L-Arg nonamer (Example 3). When coupling cycles were completed, theresin-bound peptide was treated with 20% acetic anhydride inacetronitrile, to incorporate an acetyl protecting group at theN-terminus thereof. Liberation of peptide from the resin, and theremoval of Tos groups, were achieved by treatment with hydrofluoricacid, yielding the C-terminally amidated, title compound. After removalof hydrofluoric acid, the resin/peptide mixture was washed with diethylether and extracted with aqueous acetic acid. The crude peptide waslyophilized, and then purified by RP-HPLC fractionation as described inExample 3. High resolution mass spectrometry showed the product to thedesired compound.

TAR-binding studies, performed using the mobility shift assay, indicatedthat the N- and C-protected D-Arg nonamer exhibits a TAR-bindingaffinity comparable to the basic domain of wild type tat. Bindingstudies have further revealed that this compound binds to the functionalTar molecule with specificity comparable to the wild type basic domain,relative to binding with the non-functional TAR variant. The specificityof binding has also been confirmed in studies comparing TAR binding withyeast tRNA binding.

EXAMPLE 6

Using synthesis protocols described in Example 5, to incorporate anamidated C-terminus and an acetylated N-terminus, the followingadditional oligopeptides consisting essentially of D-amino acids weresynthesized and purified for testing in the HIV inhibition assay:##STR6##

EXAMPLE 7 Inhibition of HIV Replication

Selected compounds of the present invention were tested for the abilityto inhibit HIV replication in virally infected cells of the HuT78lineage (a human cutaneous T cell lymphoma line, available from ATCCunder accession number ATCC TIB 161). Briefly, Hut78 cells (1×10⁶) weremaintained for a period of either 7 days or 14 days in growth mediumcontaining selected oligopeptide at a concentration of 5 uM. At the endof the maintenance period, the cells were washed 3 times in growthmedium and placed in triplicate T-25 flasks at a concentration of 2×10⁶cells in 5 ml of growth medium. Cells were then infected with HIV(strain NL4-3) using an inoculum of 0.1 virions/cell. After anincubation period of two hours, with mixing every 30 minutes, the virusinoculum was removed and replaced with growth medium. Four hours later,the selected oligopeptide was added to the growth medium, to a finalconcentration of 10 uM. At four and seven days after infection, samplesof culture medium were removed and tested for the presence of p24antigen, using an ELISA-based immunoassay kit available from CoulterImmunology (Hialeah, Fla., USA), in the manner instructed by thesupplier. The cultures were also monitored for HIV-induced cytopathiceffects. Controls used water in place of peptide.

Results of the assay, performed with oligopeptide 4A(D-[Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg], example 4) and witholigopeptide 4C (the D-Arg nonamer with protected ends, example 5) aregraphed in FIG. 1 (experiment 1 results). Results with these peptides,and with similarly assayed peptide 4G, i.e.,acetyl-[D-(Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg]-NH₂ (example 6) are alsotabulated below:

    ______________________________________                                               P.sub.24 Titers pg/ml                                                  Days After                                                                             24 Hrs      7 Day       14 Day                                       Infection                                                                              Pretreatment                                                                              Pretreatment                                                                              Pretreatment                                 Peptide  Day 4   Day 7   Day 4 Day 7 Day 4 Day 7                              ______________________________________                                        Expt 1                                                                        4A1                      266.3 376.4 484.8 3049                               4A2                      277.7 376.5 459.9 2662                               4C1                      168.4 100.76                                                                              152.5 175.1                              4C2                      131.5 100.72                                                                              129.0 133.6                              Control 1                356.5 892.4 690.0 3829                               Control 2                395.5 801.0 658.0 3239                               Expt 2                                                                        4C1                      98.08 48.33                                          4C2                      93.76 43.24                                          Control 1                446.50                                                                              4187.0                                         Control 2                404.10                                                                              3173.8                                         Expt 3                                                                        4G1      340.9   721.1   312.1 371.5                                          4G2      356.9   851.8   366.1 446.8                                          Control 1                                                                              473.7   1,452.8 372.1 1,348.4                                        Control 2                                                                              559.2   1,561.3 296.9 1,652.7                                        ______________________________________                                    

The results show that incubation of the cells with a 5 uM concentrationof selected oligopeptide induces significant resistance to viralinfection. At the concentration tested, peptide 4C achieves a 95% orbetter inhibition of viral replication, as measured by p24 assay (FIG.1). Visual inspection of the cells demonstrated that all selectedpeptides completely inhibited the formation of syncytia, acharacteristic of HIV-infected cells in culture. There was no apparentinhibition of host cell replication at this concentration when peptide4C was tested. In separate experiments, a 100 uM concentration ofpeptide 4C was found to have no significant detrimental effect on cellreplication, although some reduction was noted at 500 uM. This indicatesthat 4C can be formulated and used at therapeutic, non-toxic doses.

As will be seen from the above Table, peptides 4A and 4G also exhibitedpositive effects on HIV inhibition at 5 uM concentrations, particularlyfollowing pretreatment for the shorter, 7 day period.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 14                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ArgLysLysArgArgGlnArgArgArg                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note="Xaa represents a basic                          amino acid and corresponds to Y1 of the                                       specification"                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       ArgLysLysArgArgXaaArgArgArg                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note="Xaa represents a basic                          amino acid and corresponds to Y2 of the                                       specification"                                                                (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /note="Xaa represents a basic                          amino acid and corresponds to Y3 of the                                       specification"                                                                (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note="Xaa represents a basic                          amino acid and corresponds to Y4 of the                                       specification"                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ArgXaaXaaArgArgXaaArgArgArg                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note="Xaa represents Gly, Cys,                        Ser, His, Lys, Asn, or homoGln"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       TyrGlyArgLysLysArgArgXaaArgArgArgPro                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ArgGlnArgArgArgArgArgArgArg                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       ArgArgGlnArgArgArgArgArgArg                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       ArgArgArgGlnArgArgArgArgArg                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       ArgArgArgArgGlnArgArgArgArg                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       ArgArgArgArgArgGlnArgArgArg                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      ArgArgArgArgArgArgGlnArgArg                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      ArgArgArgArgArgArgArgGlnArg                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      ArgArgArgArgArgArgArgArgArgArgArgArg                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid;                                       (A) DESCRIPTION: Synthetic RNA                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GGAGAUCUGAGCCUGGGAGCUCUCUCC27                                                 (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid;                                       (A) DESCRIPTION: Synthetic RNA                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      GGAGAACUGAGCCUGGGAGCUCUCUCC27                                                 __________________________________________________________________________

We claim:
 1. A compound of the formula

    R1--(X)--R2

wherein: R1 is H, lower alkanoyl or a deaminated amino acid; R2 is OH,lower alkyl, amino, N-(loweralkyl)amino, N,N-di-(loweralkyl)amino or adecarboxylated amino acid; and X represents a D-arginine homopolymerconsisting of from 7 to 12 D-arginine residues.
 2. A compound of theformula

    Np--(X)--Cp

wherein Np represents a conventional N-terminal protecting group; Cprepresents a conventional C-terminal protecting group; and X representsa D-arginine homopolymer consisting of from 7 to 12 D-arginine residues.3. A compound according to claim 2, wherein X represents a D-argininehomopolymer consisting of from 8 to 10 D-arginines.
 4. A compoundaccording to claim 2, wherein X represents a D-arginine homopolymerconsisting of nine D-arginines.
 5. The compound acetyl-[D-(Arg)₉ ]-NH₂.6. A compound according to claim 1, wherein R1 is acetyl.
 7. A compoundaccording to claim 1, wherein R2 is amino.
 8. A compound according toclaim 1, wherein R1 is acetyl and R2 is amino.
 9. A compound accordingto claim 1, wherein X represents a D-arginine homopolymer consisting offrom 8 to 10 D-arginine residues.
 10. A compound according to claim 9,wherein R1 is acetyl.
 11. A compound according to claim 9, wherein R2 isamino.
 12. A compound according to claim 9, wherein R1 is acetyl and R2is amino.
 13. A compound according to claim 9, wherein X represents aD-arginine homopolymer consisting of 9 D-arginine residues.
 14. Acompound according to claim 13, wherein R1 is acetyl.
 15. A compoundaccording to claim 13, wherein R2 is amino.
 16. An anti-viralcomposition comprising a sterile, non-toxic carrier and a compoundaccording to claim 1 in an amount effective to inhibit viralpropagation.
 17. An anti-viral composition comprising a sterile,non-toxic carrier and a compound according to claim 2 in an amounteffective to inhibit viral propagation.
 18. An anti-viral compositioncomprising a sterile, non-toxic carrier and a compound according toclaim 3 in an amount effective to inhibit viral propagation.
 19. Ananti-viral composition comprising a sterile, non-toxic carrier and acompound according to claim 4 in an amount effective to inhibit viralpropagation.
 20. An anti-viral composition comprising a sterile,non-toxic carrier and a compound according to claim 5 in an amounteffective to inhibit viral propagation.
 21. A compound according toclaim 1, wherein R1 is H and R2 is OH.
 22. A compound according to claim21, wherein X represents a D-arginine homopolymer consisting of from 8to 10 D-arginine.
 23. A compound according to claim 22, wherein Xrepresents a D-arginine homopolymer consisting of nine D-arginines. 24.A compound of the formula

    R1--[X]--R2

wherein R1 is H, lower alkanoyl or a deaminated amino acid analog; R2 isOH, lower alkyl, amino, N-(loweralkyl)amino, N,N-di-(loweralkyl)amino ora decarboxylated amino acid; and X represents a D-arginine homopolymerconsisting of from 7 to 12 D-arginine residues.
 25. A compound accordingto claim 24, wherein X represents a D-arginine homopolymer consisting offrom 8 to 10 D-arginine residues.
 26. A compound according to claim 25,wherein X represents a D-arginine homopolymer consisting of nineD-arginine residues.
 27. An anti-viral composition comprising a sterile,non-toxic carrier and a compound according to claim 24, in an amounteffective to inhibit viral propagation.
 28. A composition for inhibitingHIV replication comprising a carrier and an anti-HIV amount of acompound according to claim 1.